Summary Aptamers (oligonucleotide-based receptors) are potentially general go-to receptors for precise and accurate, yet rapid, determination of small molecules in clinical chemistry. The systematic improvement of methods to develop high-quality aptamers for small molecules recently led to isolation of multiple outstanding aptamers that are currently being integrated in the next generation of tests for at home, real-time, and clinical laboratory (automatized, parallel) analysis. For example, at Columbia University Medical Center (CUMC), we are at a critical junction with original aptamers for phenyl alanine, vancomycin, tobramycin, and urea, all being in the process of validation on actual patients? samples in the context of their role in assays for clinical decision- making. Further, our collaborators are testing fourth generation cocaine aptamers in conjunction with our published serotonin aptamers in animal models of addiction. Despite these advances, and with over 100 successful selections behind us, there were important individual targets and even whole classes of targets, for which multiple attempts to isolate aptamers with even minimal affinity, failed unexpectedly. We now present what we learned from these failures and describe a systematic approach to isolate clinically useful aptamers for even the most challenging targets. In the process, we introduce conceptually new selections, including ?isostere-?, ?analog-?, and ?bait-and-switch? selections. . Through three Aims, we will develop algorithms for selection of clinically useful aptamers to challenging analytes. We will pursue: (1) Complex targets with solubility below that needed to capture even low-affinity aptamers (voriconazole and 25-OH vitamin D3); (2) Anionic targets with low binding free energy to aptamers isolated from small oligonucleotide libraries (valproic acid); and (3) Immunosuppressants that require either large, or extended and shallow binding pockets (cyclosporine and sirolimus, its analog everolimus, all hydrophobic, large, and with poor epitopes). Newly isolated aptamers will be continuously optimized to reach affinities required for clinical testing, turned into appropriate sensor formats, and validated as if we are preparing a laboratory-developed test (LDT) for submission to the NYS Department of Health. Beyond specific receptors for high-value targets, the important outcome of this grant will not be individual rapid procedures as it had been standard in the field, but algorithms for multistep implementation of protocols. The substantial increase in effort over standard practice is fully justified by solving widely recognized problems in clinical chemistry: enabling quantification of analytes for which there are no rapid assays with sufficient accuracy and precision for clinical decision making and frequent at ?point-of-need? monitoring.